Molecular Biology Techniques

A Classroom Laboratory Manual

Heather B. Miller, D. Scott Witherow, Sue Carson

• Publisher: Academic Press
• Year: 2011
• Edition: 3
• eBook ISBN:
  9780123855459
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

This manual is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein. The third edition has been completely re-written, with new laboratory exercises and all new illustrations and text, designed for a typical 15-week semester, rather than a 4-week intensive course. The "project approach to experiments was maintained: students still follow a cloning project through to completion, culminating in the purification of recombinant protein. It takes advantage of the enhanced green fluorescent protein - students can actually visualize positive clones following IPTG induction. - Cover basic concepts and techniques used in molecular biology research labs - Student-tested labs proven successful in a real classroom laboratories - Exercises simulate a cloning project that would be performed in a real research lab - "Project" approach to experiments gives students an overview of the entire process - Prep-list appendix contains necessary recipes and catalog numbers, providing staff with detailed instructions

Part 1. Manipulation of DNA

The goal of these laboratory exercises is to fuse a jellyfish gene with a bacterial gene and to express a single protein from this hybrid DNA sequence. Why would you want to do this? Molecular shuffling of genetic sequences, or gene cloning, is a powerful tool for understanding biological processes and for biotechnological applications. Using basic tools developed in Escherichia coli, we can ask questions about other, more complicated organisms.

Scientists have exploited E. coli both as a workhorse for producing DNA and as a source of well-characterized sequences to direct transcription and translation of foreign DNA into protein. With genetic sequence information being produced at a breathtaking rate, the limiting factor is not in sequencing DNA, but in our understanding of the function of the products of these sequences.

In terms of practical biotechnology applications, it can be a huge advantage to clone the gene encoding a difficult-to-purify protein into E. coli so that the purification process can be accomplished less expensively and to a greater degree of purity (and oftentimes more ethically, especially if a human gene is involved!). The first recombinant protein to be produced and marketed was human insulin in the early 1980s, which has been invaluable to countless diabetics. The basic tools you will learn in this class will enable you to clone, express and purify recombinant proteins. They will enable you to begin to probe the function of any protein for which a gene has been identified, and will give you the conceptual background needed for tackling more advanced techniques.

Other hosts are now commonly used for cloning DNA and expressing recombinant proteins, such as members of the bacterial genus Bacillus, as well as eukaryotic hosts including numerous species of yeast and other fungi, plants, insect cell culture, mammalian cell culture and even whole, live mammals (“pharming”). Many of the recombinant DNA methods used in this course are applicable to cloning in other hosts.

The gene we will be cloning and expressing is the enhanced green fluorescent protein gene, egfp. egfp is a brightness-enhanced variant of the green fluorescent protein from the jellyfish Aequoria victoria. ¹ The gene encoding the green fluorescent protein (and its variants, including egfp) is widely used as a “reporter gene” or “marker.” A reporter gene is a gene that is used to track protein expression. It must have phenotypic expression that is easy to monitor and can be used to study promoter activity or protein localization in different environmental conditions, different tissues, or different developmental stages. Recombinant DNA constructs are made in which the reporter gene is fused to a promoter region of interest and the construct is transformed or transfected into a host cell or organism. EGFP can also be used to mark (or tag) other proteins by creating recombinant DNA constructs that express fusion proteins that fluoresce and can be tracked in living cells or organisms. In this project, we are not using egfp as a reporter, but rather as a convenient gene to clone and assay for, as we learn the basic techniques of recombinant DNA manipulation and protein expression.

Reference

1. Yang, T; Cheng, L; Kain, SR, Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein, Nucl. Acids Res. 24 (22) (1996) 4592–4594.

Lab Session 1. Getting Oriented

Practicing with Micropipettes

Goal: Starting next week, you will be working on a laboratory project that will build throughout the entire semester. Before embarking on that journey, it is important to familiarize yourself with your lab space and to master the use of the workhorses of the molecular biology lab: the micropipettes. If your instructor has not given safety orientation yet, he or she will do so today.

Station Checklist

It is important to familiarize yourself with the work environment and laboratory equipment before beginning experiments. If the laboratory space which you are working in is shared by other laboratory sections at different times, much of the equipment can be shared. There are certain items, however, such as buffers and sterile disposable items that should not be shared between lab groups. Take a moment to go through your bench, shelves and drawers to identify equipment and reagents. Use the station checklist below and notify your instructor if anything is missing from your station. Items that are indicated as “per group” should not be shared between different sets of students on different lab days. Label these items with your initials, lab day and station number. Other items should have the station number only.

Station Checklist

Station Number ____

Name_____________________ Name________________________

_____ one power supply box

_____ one horizontal DNA minigel apparatus for agarose gels

_____ four micropipettes: P10, P20, P200 and P1000

_____ one box 1000μl sterile tips per group

_____ one box 200μl sterile tips per group

_____ one box 10μl sterile tips per group

_____ one ice bucket (or cooler or styrofoam box for ice)

_____ one box Kimwipes (Kimberly-Clark, Roswell, GA)

_____ one 15ml and one 50ml styrofoam test tube rack

_____ one pack sterile snap-cap tubes (17×100mm) for overnight bacterial cultures

_____ one test tube rack for snap-cap tubes

_____ one autoclaved container of 1.7ml microcentrifuge tubes per group

_____ two microcentrifuge tube racks

_____ one pack disposable 10ml pipettes

_____ one plastic (or electric) pipette pump

_____ one 50ml graduated cylinder

_____ one 500ml graduated cylinder

_____ one 2 liter polypropylene beaker

_____ two 1 liter polypropylene bottles, one for distilled water and one for 1X TBE buffer per group

_____ one 250 or 500ml Pyrex orange-capped bottle for melting agarose per group

_____ one thermal glove for handling microwaved agarose

_____ labeling tape

_____ permanent ink marker (Sharpie)

_____ one plastic squeeze bottle for 70% ethanol

_____ one plastic squeeze bottle for distilled water

_____ one ring-stand with clamp

_____ one pair blunt-...

Table of contents

• Cover image
• Table of Contents
• Front-matter
• Copyright
• Preface
• About the Authors
• Acknowledgements
• Note to Instructors
• Instrumentation
• Nomenclature
• Introduction
• Part 1. Manipulation of DNA
• Lab Session 1. Getting Oriented
• Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA
• Lab Session 3. PCR Amplification of egfp and Completion of Vector Preparation
• Lab Session 4. Preparation of Insert DNA (egfp) PCR Product
• Lab Session 5. DNA Ligation and Transformation of Escherichia coli
• Part 2. Screening Transformants
• Lab Session 6. Colony Hybridization
• Lab Session 7. Characterization of Recombinant Clones
• Lab Session 8. Characterization of Recombinant Clones
• Lab Session 9. Characterization of Recombinant Clones
• Lab Session 10. Computational Analysis of DNA Sequence from a Positive Clone
• Part 3. Expression, Detection and Purification of Recombinant Proteins from Bacteria
• Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot
• Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot
• Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column
• Lab Session 14. Analysis of Purification Fractions
• Part 4. Analysis of mRNA Levels
• Lab Session 15. Total RNA Purification
• Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR
• Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR
• Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR
• Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR
• Appendix 1. Equipment
• Appendix 2. Prep List
• Appendix 3. Preparation of Competent E. coli Cells
• Appendix 4. Pre-Lab Questions
• Index